U.S. patent number 5,160,486 [Application Number 07/450,557] was granted by the patent office on 1992-11-03 for test carrier utilizing reaction of two bioaffine binding partners.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Rolf Lerch, Dieter Mangold, Reiner Schlipfenbacher, Joachim Steinbiss.
United States Patent |
5,160,486 |
Schlipfenbacher , et
al. |
November 3, 1992 |
**Please see images for:
( Certificate of Correction ) ** |
Test carrier utilizing reaction of two bioaffine binding
partners
Abstract
Test carrier for analysis of a sample liquid with the help of a
specific binding reaction of two bioaffine binding partners, one of
which is contained in the sample and one in the reagent system of
the test carrier, with several capillary-active test zones (11-16;
21-25) arranged substantially next to one another on a base layer
(2). The test zones are in liquid contact with one another so that
they form a liquid transport path (20, 30) along which a liquid
flows, driven by capillary forces, from a start zone (11, 21), a
reaction thereby taking place between the first binding partner and
the reagent system containing the second reaction partner, which
reaction leads to a labelled species characteristic for the desired
analysis. A third binding partner specific for the first binding
partner sets as capturing reagent which is so arranged in a zone of
the liquid transport path (20, 30) and is so measured with regard
to the amount used that it binds a part of the first binding
partner and thereby removes it from the reaction leading to the
characteristic labelled species. A process for the analysis of a
liquid sample with the above test carrier is also provided.
Inventors: |
Schlipfenbacher; Reiner
(Lampertheim, DE), Mangold; Dieter (Maxdorf,
DE), Lerch; Rolf (Ilvesheim, DE),
Steinbiss; Joachim (Lorsch, DE) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim, DE)
|
Family
ID: |
6369521 |
Appl.
No.: |
07/450,557 |
Filed: |
December 14, 1989 |
Foreign Application Priority Data
|
|
|
|
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Dec 19, 1988 [DE] |
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3842702 |
|
Current U.S.
Class: |
422/412; 436/169;
435/287.2; 435/287.8; 435/805 |
Current CPC
Class: |
G01N
33/54386 (20130101); G01N 33/558 (20130101); Y10S
435/805 (20130101) |
Current International
Class: |
G01N
33/558 (20060101); G01N 33/543 (20060101); G01N
021/77 (); C12M 001/40 () |
Field of
Search: |
;422/56,58 ;436/169
;435/288,805 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0201339 |
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Nov 1986 |
|
EP |
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0225054 |
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Jun 1987 |
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EP |
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0271204 |
|
Jun 1988 |
|
EP |
|
0284232 |
|
Sep 1988 |
|
EP |
|
0303110 |
|
Feb 1989 |
|
EP |
|
8604683 |
|
Aug 1986 |
|
WO |
|
8906791 |
|
Jul 1989 |
|
WO |
|
Primary Examiner: Warden; Robert J.
Assistant Examiner: Blythe; Stephanie
Attorney, Agent or Firm: Felfe & Lynch
Claims
We claim:
1. Test carrier for analysis of a sample liquid by means of a
binding reaction between a first binding partner contained in the
sample and a second binding partner contained in the test carrier,
said reaction leading to a labelled species characteristic of the
desired analysis, said test carrier comprising
a base layer,
a plurality of capillary action test zones arranged on said base
layer in liquid contact with one another so as to form a liquid
transport path parallel to said base layer, said zones comprising,
in order, a sample application zone to which said sample liquid
including a first specific binding partner is applied, a conjugate
zone containing a labelled conjugate of one of said first and a
second specific binding partners, and a detection zone containing a
carrier fixed specific binding partner selected from the group
consisting of the other of said first and second specific binding
partners and a further specific binding partner which binds with
said first specific binding partner, said reaction occurring in one
of said conjugate zone and said detection zone, said labelled
species being detected in said detection zone, and
a third specific binding partner which specifically binds with said
first binding partner, said third binding partner being contained
in one of said conjugate zone and a zone between said sample
application zone and said conjugate zone, said third binding
partner being so measured that it binds only a part of the first
binding partner, thereby acting as a capturing reagent which
removes said part from the reaction leading to the characteristic
labelled species.
2. Test carrier according to claim 1, wherein the capturing reagent
is soluble in the sample liquid.
3. Test carrier according to claim 1, wherein said conjugate is a
labelled conjugate of the second binding partner and is soluble in
the sample liquid.
4. Test carrier according to claim 3, wherein the capturing reagent
is arranged in the conjugate zone.
5. Test carrier according to claim 3, wherein the conjugate zone
has a smaller flow cross-section than the next following zone in
the liquid transport path.
6. Test carrier according to claim 3 further comprising a fixing
zone in the liquid transport path after the conjugate zone, which
fixing zone contains, in carrier-fixed form, a binding partner of
the second binding partner which binds to the second binding
partner in a manner analogous to the binding of the first binding
partner to the second binding partner.
7. Test carrier according to claim 6, wherein the fixing zone has a
larger flow cross-section than the conjugate zone.
8. Test carrier according to claim 1 further comprising an
auxiliary reagent zone in which the capturing reagent is
carrier-fixed in the liquid transport path between the sample
application zone and the conjugate zone.
9. Test carrier according to claim 1, wherein the second binding
partner and the third binding partner are the same.
10. Test carrier as in claim 1 wherein said labelled conjugate is a
conjugate of said first binding partner, said second binding
partner being carrier-fixed in the liquid transport path after said
conjugate zone, whereby said first binding partner and said
labelled conjugate compete for binding to said second binding
partner.
11. Test carrier as in claim 1 further comprising a start layer in
said sample application zone, said start layer having the capacity
to take up sufficient liquid to fill the entire liquid transport
path and to pass said liquid into said zones forming said transport
path.
12. Test carrier as in claim 11 wherein said start layer comprises
a non-swelling fiber fleece bound with a binding agent which is
insoluble in the sample liquid.
13. Test carrier as in claim 11 wherein said start zone comprises a
non-swelling fiber fleece with a water-soluble binding agent.
14. Test carrier for the analytical investigation of a sample
liquid by means of an analysis reaction which includes a specific
binding reaction of a first specific binding partner which is
contained in the sample and a second specific binding partner which
is contained in the test carrier and leads to a labelled species
characteristic for the desired analysis, comprising
a plurality of capillary action test zones arranged next to one
another on a base layer and in liquid contact with one another so
as to form a liquid transport path parallel to the base layer along
which a liquid flows driven by capillary forces for performing said
analysis reaction,
said capillary action test zones including, in order, a sample
application zone to which said sample liquid including a first
specific binding partner is applied, a conjugate zone containing a
labelled conjugate of one of said first and a second specific
binding partners, and a detection zone, containing a carrier fixed
specific binding partner selected from the group consisting of the
other of said first and second specific binding partners and a
further specific binding partner which binds with said first
specific binding partner, said reaction occurring in one of said
conjugate zone and said detection zone, said labelled species being
detected in said detection zone,
said capillary action test zones comprising a first zone in said
transport path, said first zone comprising a start layer for
dipping into a sample liquid to spontaneously take up sufficient
liquid in order to fill the whole of the liquid transport path and
for passing on said liquid into remaining said zones forming said
liquid transport path,
said start layer being made of a non-swelling fiber fleece with a
binding agent insoluble in said sample liquid.
15. Test carrier according to claim 14, wherein the fibre fleece
has a retention for the sample liquid of at most 25%.
16. Test carrier according to claim 15, wherein the fibre fleece
contains at least 50% of synthetic fibres.
17. Test carrier according to claim 16, wherein the
synthetic fibres are polyamide or polyester fibres.
18. Test carrier according to claim 16 wherein the fiber fleece
contains polyvinyl alcohol.
Description
BACKGROUND OF THE INVENTION
The present invention is concerned with a test carrier for the
analytical investigation of a sample liquid by means of a specific
binding reaction of two specific binding partners, one of which is
contained in the sample and the other in the reagent system of the
test carrier. The test carrier has several capillary-active test
zones arranged substantially next to one another on a test layer,
which are in liquid contact with one another so that they form a
liquid transport path along which a liquid flows by capillary
forces from a starting zone to an end zone, a reaction thereby
taking place between the first binding partner and the reagent
system containing the second binding partner which results in a
labelled species characteristic for the desired analysis, the
labelled specific species being detected in a detection zone.
For the qualitative and quantitative analytical determinations in
the scope of the diagnosis of diseases, so-called carrier-bound
tests have recently been increasingly used. In the case of these
tests, reagents are embedded in appropriate layers of a solid test
carrier which are brought into contact with the sample. The sample
is usually a body fluid, such as blood or urine. However, it can
also be a liquid obtained by a preceding test step.
Test carriers are known in various forms. The present invention is
concerned with those test carriers in which capillary-active test
zones, which usually consist of absorbent material layers, for
example, papers, fleece or porous synthetic resin layers, are
arranged next to one another on a base layer in such a manner that
the liquid flows along the liquid path parallel to the base layer.
Therefore, these test carriers can also be referred to as "test
carriers with longitudinal transport".
Such test carrier constructions are especially advantageous for
analysis processes which are based on a specific binding reaction
of two bioaffine binding partners. Examples therefor are described
in Federal Republic of Germany patent specification No. 34 45 816,
to which U.S. Pat. No. 4,861,711 corresponds and in U.S. Pat. No.
4,361,537. Specific binding reactions in this sense are, in
particular, immunological interactions, thus reactions between
antigens or haptens, on the one hand, and antibodies on the other
hand. However, other specific interactions can also be used, such
as lectin-sugar or an active material-receptor interaction. In the
following, without limitation of the generality, reference is made
by way of example to immunological reactions.
Greatly varying test principles can be used which are described in
the literature, for example in the two above-mentioned patent
specifications.
In a first group of immunological tests, one of the front zones of
the liquid transport path contains the first binding partner
(analyte) in soluble, labelled form. A test zone provided in the
further course of the liquid transport path contains the second
binding partner in carrier-fixed form. The analyte from the sample
and the labelled analyte from the test compete for the binding
positions on the second binding partner. Therefore, such tests are
referred to as competitive tests.
In the case of a second group of known immunological tests, one of
the front zones of the liquid transport path contains the second
binding partner in soluble and labelled form. Due to the specific
binding reaction with the first binding partner, mobile and
labelled complexes are formed.
In the further course of the test, a further binding partner can be
present in carrier-fixed form which is specifically bindable with a
binding site of the first or second binding partner not saturated
by the complex formation, a sandwich thereby resulting of at least
three binding partners. Therefore, such tests are also referred to
as sandwich tests.
In the case of the so-called immunoenzymometric (IEMA) principle,
the further course of the test contains the analytes in
carrier-fixed form, the noncomplexed part of the second binding
partner thereby being fixed. Only the complexes remain mobile and
can be detected.
It is common to all immunological determinations that the analysis
reaction, which inter alia includes a specific binding reaction
between the two binding partners, leads to a labelled species
characteristic for the desired analysis. In the case of the
competitive test, this is the analyte which is bound or which
remains free. In the case of the sandwich test, these are the bound
sandwich complexes. In the case of the IEMA test, it is the complex
which remains freely mobile.
More detailed explanations of the various known immunological test
principles are not necessary because they can be found in the
appropriate literature. Independently of the special course of the
test, the present invention can be advantageously used but it is,
nevertheless, specially directed towards test carriers which work
according to IEMA principle.
Various immunological processes of determination also differ with
regard to the label employed. The present invention is especially
concerned with enzyme immune tests in which an enzyme label is
used. The labelling enzyme is usually detected by the
colour-forming reaction of a substrate of the labelling enzyme. The
substrate can, depending upon the carrying out of the test, already
be present in the detection zone or can be added thereto. In
principle, the present invention can also be used for non-enzymatic
processes in which, for example, a coloured material or a
radioactive element is used for the labelling.
SUMMARY OF THE INVENTION
In order to make available an easily handled test carrier which is
economic to produce and which is especially suitable for the
evaluation of comparatively highly concentrated analytes, for which
immunological test carriers were previously not available, the
present invention provides a test carrier for the analytical
investigation of a sample liquid with the help of a specific
binding reaction of two binding partners, of which a first is
contained in the sample and a second is contained in the reagent
system of the test carrier. Several capillary-active test zones are
arranged substantially next to one another on a base layer, which
test zones are in liquid contact with one another so that they form
a liquid transport path along which a liquid flows, driven by
capillary forces, from a start zone. A reaction thereby takes place
between the first binding partner and the reagent system containing
the second binding partner, which reaction leads to a labelled
species characteristic for the desired analysis. A specific third
binding partner acting as capturing reagent for the first binding
partner is so arranged in a zone of the liquid transport path and
is so measured with regard to the amount used that it binds a part
of the first binding partner and thereby removes it from the
exchange action leading to the characteristic labelled species.
Immunological determinations are characterised by an extremely high
sensitivity. This is an advantage for low concentrated analytes but
endeavours have long since been made also to use the advantages of
immunological determinations for higher concentrated analytes. The
great sensitivity thereby proves to be a serious problem. Of
course, it is possible manually to dilute the sample
correspondingly. However, this requires a handling step which can
only be carried out by trained laboratory personnel and which is
time-consuming. Therefore, attempts have been made to reduce the
sensitivity of the test process by laborious means. For example,
binding partners are used in the test which display a reduced
affinity towards the analyte or the stoichiometry between the
labelled binding partner and the labelling enzyme is
manipulated.
SUMMARY OF THE INVENTION
In comparison with these known, laborious processes, the present
invention now provides a surprisingly simple way by providing a
capturing reagent specific for the analyte. A reduction of the
analyte concentration on the test carrier is thereby to some extent
achieved without a separate previously provided dilution step being
necessary. The carrier according to the present invention is
advantageous for concentrations of above 10.sup.-8 mole/liter and
especially of above 10.sup.-7 mole/liter.
A further proposal which represents a valuable supplementation of
the above-mentioned measures but, nevertheless, also has an
independent importance, relates to the starting zone, the
construction of which is important for the handling and function of
the test carriers, which is explained in more detail in the
following.
In this regard, the present invention proposes, in the case of a
test carrier of the initially mentioned type, the production of the
starting zone from a non-swelling fibre fleece which contains a
binder which is insoluble at ambient temperature.
Also in this regard, the present invention differs fundamentally
from the prior art. Thus, in Federal Republic of Germany patent
specification No. 34 45 816 and in European patent specification
No. 0,052,328, it is stated that especially synthetic resin sponges
or layers of hydrophilic materials are suitable which, on the basis
of their swelling properties, have an especially high water
take-up. It is thereby ensured that the starting zone, in the case
of dipping into a sample liquid, takes up sufficient liquid in
order to fill the whole of the liquid transport path of the test
carrier, even when, after the dipping into the sample, no further
liquid is supplied.
Surprisingly, we have found, in the scope of the present invention,
that especially good results can be achieved with a comparatively
hydrophobic material, which, referred to its inherent weight, has a
smaller liquid take-up but is emptied to a high degree on the test
carrier.
Quantitatively, this property can be expressed as liquid retention.
In the scope of the present invention, this is determined by laying
a sample of 25 cm.sup.2 surface area on a sponge cloth which is
considerably larger than the sample and is wetted through to
saturation. The amount of water removed from the sponge is
determined by weighing. The wet sample is laid on a round filter of
the type 2668/8 of the firm Schleicher & Schull, Dassel,
Federal Republic of Germany, with a diameter of 158 mm. and removed
after 2 minutes. The amount of water taken up by the round filter
is again determined by weighing. The retention is determined as the
percentage relationship of the amount of water remaining in the
sample to the amount of water originally taken up. In this sense,
materials are preferred which have a liquid retention of at most
25% for the sample liquid.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a schematic view illustrating the principle of the zone
sequence in a test carrier; and
FIG. 2 is a perspective view of the test carrier.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
FIG. 1 serves for the explanation of the various possible test
principles and arrangements of the capturing reagent of the test
carrier. Therefore, it omits constructional details and merely
shows, in principle, a test carrier 1 with a base layer 2 on which
are arranged next to one another, with their end faces abutting,
test zones 11 to 16. The test zones preferably each consist of
various absorbent materials (papers, fleece, porous synthetic resin
layers and the like), the liquid contact on the abutting edges
being achieved by a sufficiently close placing together of the
layers. However, it can also be advantageous for the layers partly
to overlap (cf. FIG. 2) or for several adjacent zones to be
produced in one piece from the same material. In all, the test
zones form a liquid transport path 20 which runs from the start
zone 11 to the end zone 16.
In the case of the use of the test carrier, at first only the start
zone 11 is contact with the sample liquid, preferably by
appropriately dipping the test carrier into the sample liquid.
Thereafter, the start zone gradually gives up its liquid content to
the liquid transport path. The liquid chromatographs mainly driven
by the capillary forces prevailing in the zones 11 to 16 up to the
end zone 16. Details of the thereby important liquid transport
properties of the zones are dealt with in more detail in connection
with FIG. 2.
The start zone 11 is followed in the illustrated case by a sample
application zone 12 and an auxiliary reagent zone 13. The function
of the sample application zone 12 is explained in more detail
hereinafter. The auxiliary reagent zone 13 contains a buffer in
order to adjust the pH value of the sample to the requirements of
the test. If desired, it can contain further auxiliary reagents,
for example wetting agents and the like. In general, there can be
present more or less zones than illustrated, depending upon the
requirements of the test.
Depending upon the individual case of the immunological test
principle to be used, immunological components of the reagent
system are contained in the zones of the test carrier. Some
examples are explained in the following.
In the case of a competitive test for the determination of an
antigen Ag contained in the sample, the zone 14 can, for example,
contain a conjugate AgE of the antigen with a labelling enzyme in
soluble form. The zone 15 contains, as second binding partner, an
antibody in carrier fixed form Ab1(f).
In the case of flow through of the sample, the AgE is dissolved. Ag
and AgE compete for binding positions on the Ab1(f). When the
liquid further flows into the end zone 16, a separation takes place
between the AgE fixed in the zone 15 by binding on the Ab1(f) and
the AgE remaining free, which flows further into the zone 16. The
amount of AgE bound in the zone 15 is specific and characteristic
for the analysis and can be detected there in that, for example, in
a further process step, a colour-forming substrate is supplied to
the enzyme and the colour formation in the zone 15 is observed. In
this regard, the course of the test is conventional (cf., for
example, U.S. Pat. No. 4,861,711.
As capturing reagent, there is present in the zone 13 a
carrier-fixed third binding partner Ab2(f). It serves the purpose
of fixing a part of the sample antigen before it passes into the
zone 14 with the antigen conjugate. In this way, a certain part of
the antigen (thus, generally expressed, the first binding partner)
is removed from the analytical reaction leading to the labelled
species.
As mentioned, the antibodies Ab1(f) and Ab2(f) must, in each case,
be specifically bindable with the same antigen. The same antibody
can preferably be employed.
In the case of a test carrier working according to the
immunoenzymometric test principle (IEMA) for the determination of
an antigen Ag contained in the sample, the zone 14 contains a
soluble conjugate AbE of an antibody with a labelling enzyme
(second binding partner), bind specifically with the antigen. The
zone 15 contains the antigen in carrier-fixed form (Ag(f), further
binding partner). In the case of the flowing through of the sample,
the AbE is dissolved and forms complexes Ag-AbE with the Ag.
Non-complexed AbE is bound in the zone 15 on to the Ag(f), whereas
the Ag-AbE complexes remain free. When the liquid continues to flow
into the zone 16, there takes place a separation of the bound AbE
and of the freely mobile Ag-AbE complexes characteristic for the
analysis. The latter can be detected in the zone 16, a
colour-forming substrate of the labelling enzyme thereby
advantageously being present in the zone 16. This part of the test
is also known.
Whereas in the case of the competitive test, the detection takes
place as described above preferably in the zone 15, i.e. in the
zone with the fixed binding partner, AbL(f) in the case of the
immunoenzymometric test, the zone 16 following the fixing zone 15
serves as the detection zone.
Immunoenzymometric tests are characterised by an especially high
sensitivity and a simple handling. In the scope of the present
invention, we have, surprisingly, shown that test carriers which
work according to this principle can also be successfully used for
the direct detection of comparatively highly dosed analytes
(concentrations greater than 0.1 .mu.mole/liter) without the
advantages with regard to the ease of handling thereby being
lost.
Again, a capturing reagent is provided which, just as in the case
of the previously described competitive test principle, can be
contained in the zone 13, i.e. in a zone of the test carrier placed
before the conjugate zone, in soluble or carrier-fixed form.
Another variant is especially preferred in which the capturing
reagent is present in soluble form, together with the conjugate, in
the same zone (in the case of the example thus in zone 14). In this
case, the antibody used as capturing reagent should have the same
specificity of action as the antibody used in the conjugate.
Therefore, there is especially suitable an identical monoclonal
antibody but optionally also a polyclonal antibody with a very
narrow epitope spectrum.
In the case of this variant of the present invention, the capturing
reagent, which has no enzyme labelling, competes with the conjugate
for the specific binding positions on the analyte. There is thereby
achieved an essentially percentage "dilution", i.e. the portion of
the analyte which is removed by the capturing reagent from the
detection reaction corresponds approximately to the ratio between
capturing reagent and conjugate.
The possibility of providing the capturing reagent in free form is
not limited to the case in which it is provided in the same zone as
the conjugate. In its complete generality, the present invention
encompasses, on the contrary, also test carriers in which a soluble
capturing reagent is contained in a suitable zone of the liquid
transport path before the detection zone.
The amount of the capturing reagent is to be adjusted to the
specific requirements. Theoretically, it must be smaller than the
smallest amount of the analyte in the sample to be detected (limit
of detection). In practice, however, it depends upon various
factors. These include the affinity constant of the antibody used,
details of the structure of the conjugate and the speed with which
the liquid flows through the zone which contains the capturing
reagent. Also of importance is the extent to which the binding of
the sample antigen to the capturing reagent is complete. On the
basis of theoretical considerations, one should have expected that
this would include such considerable uncertainties that a
considerable falsification of the measurement results are to be
feared. However, in practice, we have, surprisingly, shown that the
amount of capturing reagent can be empirically so determined that
an immunological determination of highly concentrated parameters is
possible with good accuracy.
Preferably, the absorbent layer material which forms the test zone
with the capturing reagent is impregnated with an impregnation
solution in which is contained the capturing reagent in a molar
concentration which corresponds approximately to the desired limit
of detection of the analytical determination. The concentration of
the capturing reagent in the impregnation solution should be at
least half as great as the desired limit of detection. In absolute
figures, the concentration is at least 10.sup.-8 mole/liter and
preferably at least 10.sup.-7 mole/liter.
The sample need not necessarily be applied to the starting zone. On
the contrary, a special sample application zone 12 can be provided.
This is, in particular, advantageous when an additional sample
dilution is to be achieved.
The sample is thereby applied in insufficiency to the sample
application zone 12. "In insufficiency" is to be understood, in
this case, that the volume applied is smaller than the absorbent
capacity of the sample application zone. Subsequently, the start
zone is contacted with an elution agent, which is advantageously
water or a buffer solution.
We have found that a dilution can thereby be achieved. The degree
of the dilution can be influenced by the volume pipetted on to the
sample application zone and the absorptive capacity of the start
zone. An especial advantage of this method of handling is the fact
that the samples can be dosed on at any desired point of time and
allowed to dry, the elution and analyses then first taking place at
a later point of time. In this way, series of test strips can be
collected.
It is important for the dilution function of the sample application
field that no enrichment of the sample disturbing the analysis
occurs in the front of the chromatographing liquid. For this
reason, it is advantageous to select for the sample application
zone a material which fulfils this requirement on the basis of its
particular properties. To these belong the following working
principles:
a) The material is so chosen that the analyte pipetted on binds
weakly on the solid phase and is retardedly eluted by the elution
agent.
b) The material of the sample application zone is so structured
that, by eddying and/or non-laminar flow, a mixing of the sample
and elution agent is achieved.
FIG. 2 shows a test carrier which, in an especially simple way,
permits the determination of comparatively highly dosed parameters
with an immunoenzymometric test principle. It is especially
suitable for the determination of such parameters in a sample
liquid which is available in comparatively large amounts,
especially in urine.
On a base layer 2 are arranged essentially next to one another a
start zone 21, an auxiliary reagent zone 23, a conjugate zone 24
and a fixing zone 25. A colour-forming zone 26 is present between
the end 25a of the fixing zone and the base layer. The end 25a of
the fixing layer is thereby pressed by a holding-down layer 28 made
from a stiff synthetic resin material against the colour-forming
layer 26. The holding-down layer is fixed with a melt adhesive
strip 27 to the base layer 2.
In FIG. 2, it can be seen that the layers 21 to 25 each overlap the
neighbouring layer slightly in order to produce a better liquid
contact on the edges of the zones. Essentially, however, the liquid
transport takes place in the longitudinal direction of the test
carrier and thus parallel to the surface of the layer materials
from which the zones are produced, along the liquid transport path
30.
In use, the test carrier is dipped into the sample liquid to such
an extent that only the start zone 21 is wetted. Thus, only the
start zone makes contact with the sample liquid. Therefore, it must
be such that it takes up the liquid spontaneously and completely
and passes it on or gives it up well. For a test strip of the type
here in question, it is important that all the zones placed after
the start zone 21, which form the actual functional range of the
test carrier, are supplied sufficiently and reproducably with
liquid. This can be achieved by leaving the test carrier to stand
in a vessel with the sample so that a continuous contact is present
to a large supply of liquid. However, this makes the handling
difficult. Therefore, it is desirable that the start zone 21 takes
up a sufficient amount of liquid within a few seconds and,
according to the test requirements, again gives it off after the
test strip has been taken out of the sample liquid.
In contradistinction to the above-mentioned older suggestion
according to European patent application No. 0,052,328, according
to the present invention, the start zone consists of a nonswellable
fibre fleece with a water-insoluble binding agent.
Especially preferred as non-swelling fibre materials are fully
synthetic fibres. The fleece should contain at least 50% of such
material. Polyamides and polyesters, as well as mixtures thereof,
have proved to be especially useful.
As binding agent for the consolidation of the fibres, polyvinyl
alcohol can advantageously be used. This takes place especially in
such a manner that, in the production of the fleece, the actual
fleece fibres are mixed in a vat with fibres of polyvinyl alcohol.
They are then, as is usual in the case of the production of fleece,
spread out on a drying roller. At a drying temperature of about
90.degree. to 150.degree. C., the polyvinyl alcohol fibres dissolve
and form a coating on the other components of the fleece which
imparts stability to the fleece. There is preferably used a high
molecular weight, fully saponified polyvinyl alcohol with a
specific weight of from 1.26 to 1.30 g./cm.sup.3.
Especially preferred polyester fibres have a specific weight of
about 1.17 g./cm.sup.3, a length of cut of 6 to 12 mm. and a fibre
fineness of 1.7 to 3.3 dtex.
Insofar as polyamide fibres are used, they preferably have a
specific weight of about 1.14 to 1.15 g./cm.sup.3, a length of cut
of 4 to 6 mm. and a fibre fineness of 2.2 dtex.
As additional component, the fleece can contain linters from the
base wool of the fibres obtained from cotton plants which have been
chemically digested and bleached.
The further zones contain the same reagents as described in FIG. 1
for the case of an immunoenzymometric test. The auxiliary reagent
zone 23 contains a buffer and optionally further adjuvant reagents,
the conjugate zone 24 an enzyme conjugate of a (second) binding
partner specifically bindable with the analyte, the fixing zone 25
solid-phase-bound analyte (or analyte analogue) and the
colour-forming zone 26 a colour-forming substrate of the labelling
enzyme.
For the determination of an antigen Ag, in the conjugate AbE zone
24 is used a conjugate of a corresponding antibody and in the
fixing zone 25 the same antigen Ag or another antigen Ag* bindable
with the antibody Ab of the layer 24. If an antibody is to be
determined, then, as is known to the expert, in each case antigen
is to be replaced by antibody.
Also in agreement with FIG. 1, a capturing reagent is provided,
which is preferably soluble and identical with the enzymatically
conjugated binding partner in the layer 24.
The course of the test corresponds to the immunoenzymometric
variant of the test carrier according to FIG. 1 and, therefore,
does not have to be described again here.
For the function of the test carrier according to FIG. 2, the
liquid transport properties of the layer materials which form the
different zones is of especial importance.
For an optimum accuracy of the test, it is necessary that the
species characteristic for the analysis is present substantially
homogeneously over the whole of the surface so that a homogeneous
colour formation is obtained. Thus, in the case of an
immunoenzymometric test, a homogeneous distribution of the Ag-AbE
complexes is to be achieved in the colour-forming zone 26
characteristic for the analysis.
In the scope of the present invention, we have found that, in this
sense, it is advantageous when the materials from which the
conjugate zone and the next-following zone are produced have their
absorbent properties so adjusted to one another that the material
of the conjugate zone transports the liquid faster than the
material of the next-following zone.
In the illustrated example, this refers to the conjugate zone 24
and the next-following zone 25 which, in the most general case,
does not absolutely have to be a fixing zone. Since both zones lie
in the same liquid stream, in the stationary state, when both zones
are filled, they must transport the same amount of liquid per unit
time. Slower flow of the liquid nevertheless results since the
conjugate zone is thinner and therefore has a smaller flow
cross-section than the next following zone.
This measure promotes the homogenisation of the conjugate.
Depending upon the solution and liquid transport relationships of
the conjugate zone 24, the conjugate is there dissolved by the
entering liquid in the sense of a more or less sharp concentration
front. This corresponds to the normal behaviour of an absorbent
layer in liquid chromatography. It is the purpose of the
here-discussed measure that the concentration differences resulting
therefrom are to equalise. The conjugate zone is first filled
comparatively fast, with little dissolving of the conjugate. The
further liquid transport then follows comparatively slowly,
corresponding to the liquid transport properties of the following
zone. During this slow further flow, the conjugate already in the
zone 24 dissolves comparatively homogeneously. Furthermore, the
slow flow brings about an equilibrating effect within the zone
25.
In the case of the immunoenzymometric test, there is present, as
explained, a fixing zone 25 behind the conjugate zone 24 and, in
the most general case, further zones 24 can be present between the
two zones 25. A preferred measure of the present invention provides
that the fixing zone has a larger flow cross-section than the
conjugate zone.
In the case of known immunological tests, there are preponderantly
used fine-pore synthetic resin materials (membranes) of small
thickness as fixing layer because they permit a high loading
density of the carrier-fixed immunological reagents and the liquid
flows slowly through them. A dependable fixing of the non-complexed
conjugate from the preceding conjugate layer is thereby achieved.
This is of great importance for the accuracy of the
measurement.
Deviating from this previously known way, in the scope of the
present invention, there is now preferably used a fixing layer
which is produced on the basis of a comparatively loose carrier
material, for example paper, fabric or especially preferably
fleece. The layer material is relatively thick so that the fixing
zone has a greater flow cross-section than the conjugate zone. In
this way, there can here also be achieved a very high loading with
immune reagents, in which case the production is considerably
easier than in the case of a membrane. Due to the mentioned
balancing of conjugate zone and fixing zone, there is given at the
same time a comparatively slow flow in the fixing layer, which is
of advantage for a complete binding of the excess conjugate.
The homogeneity of the colour formation in the layer 26 is, in the
case of the illustrated test carrier, also promoted in that the
colour-forming zone runs parallel to a part of the fixing zone and
is in a laminar liquid contact with this. At the same time, the
colour-forming zone is advantageously so formed that the liquid
from the fixing zone 25 only penetrates retardedly into it or the
substrate in the zone 26 dissolves retardedly in such a way that
the fixing zone 25 has filled substantially completely before the
colour-forming reaction commences. In this regard, reference is
made to Federal Republic of Germany patent application Nos. P 38 26
056.5 and P 38 26 057.3 of the 30th Jul., 1988. These applications
correspond to respective U.S. application Ser. Nos. 384,982 and
384,726.
The following Examples are given for the purpose of illustrating
the present invention:
EXAMPLE 1
Water Take-Up and Output Properties of Materials Suitable for the
Start Zone 11,21
A swelling sponge material according to European patent application
No. 0,052,328 was compared with the following three materials
according to the present invention:
a) Mixed Fleece of Polyamide and Polyester
The materials were mixed in a vat together with Kuralon (polyvinyl
alcohol produced by Rohtex-Textil, Monchengladbach, Federal
Republic of Germany) in a weight ratio of
polyamide:polyester:Kuralon of 30:20:2.6 and worked up in known
manner to give a fleece with a weight per unit surface area of 160
g./m.sup.2 and a thickness of 1.0 mm.
b) Pure Polyester Fleece
Polyester and Kuralon were mixed in a vat in a weight ratio of 10:1
and worked up to give a fleece with a weight per unit surface area
of 166 g./m.sup.2 and a thickness of 1.0 mm.
c) Polyester Fleece with Linters
Polyester, linters and Kuralon were mixed in a vat in a weight
ratio of 50:25:7.5 and worked up to give a fleece with a weight per
unit surface area of 250 g./m.sup.2 and a thickness of 1.4 mm.
The following Table shows the retention behaviour of such
fleece:
TABLE ______________________________________ water water take-up
output retention ______________________________________ sponge
according to 5632 ml/m.sup.2 3862 ml/m.sup.2 31.4% EP 0,052,328
fleece a) 1825 ml/m.sup.2 1636 ml/m.sup.2 10.4% fleece b) 1751
ml/m.sup.2 1568 ml/m.sup.2 10.5% fleece c) 1866 ml/m.sup.2 1548
ml/m.sup.2 17.1% ______________________________________
It can be seen that the water take-up is the highest in the case of
the previously known sponge. The same also applies to the water
output. However, of especial importance for the function of the
test carrier is the retention, i.e. the percentage of the liquid
remaining behind in the particular test zone under certain defined
experimental conditions. From the example, it can be seen that this
value is, in the case of all three compositions according to the
present invention, considerably better than in the case of the
previously known start zone material.
EXAMPLE 2
Test Carrier for the Determination of Albumin in Urine and
Especially for the Detection of Microalbuminuria
A test carrier according to FIG. 2 was produced as follows:
Start Zone 11
Polyester fleece 1.110 of the firm Binzer, Hatzfeld, Federal
Republic of Germany. It is a pure polyester fleece consolidated
with 10% Kuralon. The thickness is 1.0 to 1.2 mm and absorption
capacity 1800 ml./m.sup.2.
Buffer Zone 23
A fleece material SL 4207 KA of the firm Kalff, Euskirchen, Federal
Republic of Germany, consisting of 90% polyester, 10% regenerated
cellulose and a small amount of acrylate, with a thickness of 0.7
mm. and an absorptive capacity of 480 ml./m.sup.2, was impregnated
with the following solution and subsequently dried:
200 mM sodium phosphate, pH 7.8
1% bovine serum albumin.
Conjugate Zone 24
A glass fibre fleece of 100 parts by weight of glass fibre,
consolidated with 10 parts by weight of Kuralon, in a thickness of
0.2 mm. and with an absorption capacity of 200 ml./m.sup.2, was
impregnated with the following solution and subsequently dried:
70 mM sodium phosphate, pH 7.4
1% trehalose
0.5% bovine serum albumin
6 kU/liter conjugate of .beta.-galactosidase and analyte-specific
antibody (IgG)
70 mg./l unlabelled analyte-specific antibody (IgG) as capturing
reagent.
Fixing Zone 25
A mixed fleece of polyester, cotton linters and Etadurin.RTM. in a
weight ratio of 50:50:3 with a thickness of 0.5 mm. and an
absorption capacity of 450 ml./m.sup.2 was impregnated with the
following solution and subsequently dried for 30 minutes at
50.degree. C. 10 mM sodium phosphate buffer, pH 7.5 cross-linked
human serum albumin in a concentration of
200 mg./ml. sodium phosphate buffer
The cross-linked human serum albumin was prepared as follows:
1.5 g Human serum albumin was placed in 30 ml. of 200 mM potassium
phosphate buffer (pH 8.0) and mixed within the course of 2 hours
with 2.5 ml. of a solution of 50 mg. disuccinyl suberate/ml.
dioxan. After termination of the cross-linking reaction, dialysis
was carried out against a 500 fold volume of 20 mM potassium
phosphate buffer (pH 7.2). The high molecular weight fraction with
a molecular weight of more than 650,000 Dalton was separated on
Superose 6.RTM. (Pharmacia, Freiburg, Federal Republic of Germany)
by gel filtration and, after the addition of 6 mg. saccharose/mg.
protein, lyophilised.
Colour-Forming Zone 26
On a polycarbonate film of 0.1 mm. thickness was coated a soluble
film of the following formulation:
4.5 g Mowiol 18/88 (firm Hoechst, Frankfurt am Main, Federal
Republic of Germany)
0.3 g o-nitrophenol-.beta.-galactoside dissolved in 50 ml.
water.
* * * * *